The attachment of metal ions to proteins, peptides and other, smaller molecules is a fast expanding technology, which has numerous proven and potential applications in research, in industry and, particularly, in medicine.
In recent years, much of the impetus behind the development of this technology has been the ability to link metal ions to antibodies, especially monoclonal antibodies. Such metal labelled antibodies have found a widespread use, especially in medicine, where they have been employed, for example, to target the metal ions to a specific tissue type, both in vitro and in vivo. Thus, metal labelled antibodies have applications in locating specific tissue types (e.g. employing computer-aided tomographic techniques where the metal ion is in some way detectable) and in the treatment of cell disorders (e.g. treating mammalian tumours where the metal ion is a cytotoxic radionuclide).
Conventionally, attachment of the metal ion to a protein such as an antibody has been achieved by complexation by an acyclic chelate such as a substituted diethylenetriaminepentaacetic acid [Gansow O. A. et al, Inorg. Chem., (1986), 25, 2772, and see for example, U.S. Pat. No. 4454106] or ethylenediaminetetraacetic acid [Meares, C. F. et al, Acc. Chem. Res., (1984), 17, 202] covalently linked to the antibody. Such acyclic complexes however tend to be unstable in vivo either as a result of acid-catalysed decomplexation or competitive chelate binding by Ca.sup.2+ or Zn.sup.2+ in serum, or as a result of competition from transferrin [Moerlein, S. M. et al, Int. J. Nuc. Med. Biol., (1981) 8, 277]. The lack of stability can result in uncomplexed metal atoms in the body which have a cytotoxic effect on healthy tissue (e.g. bone marrow) or which markedly reduce the signal-to-noise ratio of an imaging technique.
A possible alternative to the use of acyclic chelates in the labelling of antibodies is the use of macrocyclic ligands, which has previously been suggested in broad terms [Gansow O. A. et al, Am. Chem. Soc. Symp. Ser., (1984), 241, 215; UK Patent Specification No. 2122641; and Moi M. K. et al, Anal. Biochem., (1985), 148, 249-253]. More recently, tri-aza and tetra-aza macrocycles have been described which are capable of binding metals, and which can be conjugated to antibodies (International Patent specifications Nos. WO 89/01475 and WO89/01476).
Other tri-aza macrocycles have also been described, which are capable of binding metals [International Patent Specification No. WO86/02352; European Patent Specification No. 197437; Bryden, C. C. et al, Rare Earths Mod. Sci. Technol. (1982), 3, 53-57; Kabachnik, I. M. et al, Izv. Akad. Nauk. SSSR, Ser. Khim., 835 (1984)]. Some compounds of these types, when complexed with metals, have been proposed for use as contrast agents for use in diagnostic imaging.
We have now found a new class of functionalised tri-aza macrocycles, members of which are able to form kinetically inert complexes with metal ions which are of use in diagnosis and therapy.